The present invention relates to new types of biodegradable, terminally activated polymers which are useful in forming conjugates of bioactive materials. In particular, the invention relates to biodegradable, polymeric-based conjugates having increased therapeutic payloads and methods of preparing the same.
Over the years, several methods of administering biological y-effective materials to mammals have been proposed. Many medicinal agents are available as water-soluble salts and can be included in pharmaceutical formulations relatively easily. Problems arise when the desired medicinal agent is either insoluble in aqueous fluids or is rapidly degraded in vivo. Alkaloids are often especially difficult to solubilize.
One way to solubilize medicinal agents is to include them as part of a soluble prodrug. Prodrugs include chemical derivatives of a biologically-active parent compound which, upon administration, eventually liberate the parent compound in vivo. Prodrugs allow the artisan to modify the onset and/or duration of action of an agent in vivo and can modify the transportation, distribution or solubility of a drug in the body. Furthermore, prodrug formulations often reduce the toxicity and/or otherwise overcome difficulties encountered when administering pharmaceutical preparations. Typical examples of prodrugs include organic phosphates or esters of alcohols or thioalcohols. See Remington""s Pharmaceutical Science, 16th Ed, A. Osol, Ed. (1980), the disclosure of which is incorporated by reference herein.
Prodrugs are often biologically inert or substantially inactive forms of the parent or active compound. The rate of release of the active drug, i.e. the rate of hydrolysis, is influenced by several factors but especially by the type of bond joining the parent drug to the modifier. Care must be taken to avoid preparing prodrugs which are eliminated through the kidney or reticular endothelial system, etc. before a sufficient amount of hydrolysis of the parent compound occurs.
Incorporating a polymer as part of a prodrug system has been suggested to increase the circulating life of a drug. However, it has been determined that when only one or two polymers of less than about 10,000 daltons each are conjugated to certain biologically active substances such as alkaloid compounds, the resulting conjugates are rapidly eliminated in vivo, especially if a somewhat hydrolysis-resistant linkage is used. In fact, such conjugates are so rapidly cleared from the body that even if a hydrolysis-prone ester linkage is used, not enough of the parent molecule is regenerated in vivo to be therapeutic.
As an outgrowth of the work in the prodrug field, it has been thought that it would be beneficial in some situations to increase the payload of the polymeric transport form. This technique was offered as an alternative to the many approaches in which a single molecule of a therapeutic moiety containing a substitutable hydroxyl moiety is attached to a terminal group found on the polymer. For example, commonly-assigned PCT publication WO96/23794 describes bis-conjugates in which one equivalent of the hydroxyl-containing drug is attached to each terminal of the polymer. In spite of this advance, techniques which would further increase the payload of the polymer have been sought. In addition, technologies for forming prodrugs of therapeutic moieties having a substitutable amino group have also been sought. The present invention addresses these needs.
The present invention includes compounds of formulae (X) and (XI): 
wherein:
R31 is a linear or branched polymer residue;
Y10 and Y11 are independently O, S, or NR40;
R32-R40, R50 and R51 are independently selected from the group consisting of hydrogen, C1-6 alkyls, C3-12 branched alkyls, C3-8 cycloalkyls, C1-6 substituted alkyls, C3-8 substituted cycloalkyls, aryls, substituted aryls, aralkyls, C1-6 heteroalkyls and substituted C1-6heteroalkyls;
a, b and e are each independently selected positive integers, preferably from about 1 to about 6;
X1 and X2 are independently O, S or NR41;
wherein R41 is selected from the same group as that which defines R40;
L is an amino acid residue or a bifunctional linker.
X3 is 
wherein Y12 and Y13 are independently O, S, or NR40;
Z is selected from the group consisting of a bond, a moiety that is actively transported into a target cell, a hydrophobic moiety, and combinations thereof;
D1 and D2 are independently one of OH a residue of a hydroxyl-containing moiety, a residue of an amine-containing moiety or a leaving group; and
y1 and y2 are each independently a positive integer.
In preferred aspects of the above embodiment,
R31 preferably a PEG residue;
Y11 and Y12 are both O;
R22-R40, R50, and R51 are each hydrogen or a C1-4 alkyl;
a and b hare each 1;
y1 and y2 are both one; and
D1 and D2 are both residues of either a hydroxyl- or amine-containing moiety such as one having biological activity as defined herein.
With regard to L in formulae (X) and (XI), a non-limiting list of suitable amino acid residues include those of the formula: 
wherein X4 is O, S or NR42, Y14 is O, S, or NR45; where R42, R45 and R52-R53 are selected from the same group which defines R40; and (f) is a positive integer, preferably from about 1 to about 2. Alternatively, a non-limiting list of suitable bifunctional linkers include: 
wherein X5 is O, S or NR43;
Y15 is O, S, or NR44;
R43, R44 and R54-R58 are selected from the same group which defines R40; and g is a positive integer, preferably from about 1 to about 2.
With regard to (Z), it will be understood that in addition to being a covalent bond, Z is covalently linked to [D]y, so that Z is a moiety that is actively transported into a target cell, a hydrophobic moiety or a combination thereof. Optionally, Z is monovalent, multivalent, or more preferably, bivalent, wherein (y) is 1 or 2. Z itself optionally includes an amino acid residue, a sugar residue, a fatty acid residue, a peptide residue, a C1-18 alkyl, a substituted aryl, a heteroaryl, xe2x80x94C(xe2x95x90O), xe2x80x94C(xe2x95x90S), and xe2x80x94C(xe2x95x90NR42), where R42 is selected from the same group which defines R40.
When Z includes at least one amino acid residue, the amino acid is, e.g., alanine, valine, leucine, isoleucine, glycine, serine, threonine, methionine, cysteine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, lysine, arginine, histidine, proline, and/or a combination thereof, to name but a few. When Z includes a peptide, the peptide ranges in size, for instance, from about 2 to about 10 amino acid residues. In one preferred embodiment, the peptide is Gly-Phe-Leu-Gly or Gly-Phe-Leu.
Methods of preparing and using the same are also disclosed.
In more general aspects, the invention includes compounds of the formula:
(D)n-M-(R1)mxe2x80x83xe2x80x83(I)
wherein
(m) and (n) are independently selected positive integers, preferably from about 1 to about 6 each;
D is a residue of a biologically active moiety;
M is a multifunctional linker/spacer moiety; and
R1 is a polymer residue.
With respect to the linking of the polymer strands, the artisan is provided with higher total molecular weight polymers which are useful in providing therapeutic conjugates with relatively long T1/2""s. There are several advantages associated with these types of polymers. For example, depending upon the linkages used to attach the polymer strands to the multifunctional spacer groups, the artisan can design relatively high molecular weight polymeric transport systems which will predictably biodegrade into polymers of relatively low molecular weight which are more readily eliminated from the body than the singular polymer of higher molecular weight. Secondly, because relatively small molecular weight polymers are used to build the biodegradable transport form, the polydispersity associated with some single strand high molecular polymers such as when PEG has a molecular weight of over 40 kDa is substantially avoided.
Additional advantages associated with using the multifunctional spacers in the polymers of the present invention are that there is a large number of multifunctional moieties readily available. Thus, the artisan can prepare polymeric prodrug systems having high degrees of loading, i.e. 3-6 or more molecules of active drug per transport system. A still further advantage of the multifunctional spacers of polymeric systems of the present invention is that the artisan can form prodrugs of almost any therapeutic molecule. For example, the multifunctional spacer can be designed to include the capability of accommodating a linker for attaching to hydroxyl residues, amine residues, sulfhydral residues, etc. found on organic molecules, proteins, peptides, enzymes, etc. Another advantage is that the linkers can be selected to achieve a proper balance between the rate of parent drug-polymer linkage hydrolysis on the one hand and the rate of clearance of prodrug from the body on the other which is caused by lower molecular weight polymer portions being released from the transport system. The linkages between the polymer and the parent compounds, also referred to herein as biologically-active nucleophiles, hydrolyze at a rate which allows a sufficient amount of the parent molecules to be released in vivo before clearance of the prodrug from the plasma or body.
The high payload polymeric conjugates of the present invention are thus unique delivery systems which can contain up to several molecules of a drug per unit.
In the case of compounds corresponding to formula (X), the artisan is provided with a unique polymer transport form which can deliver more than one equivalent of biologically active material using a backbone which includes multiple strands of polymer. In the case of compounds corresponding to Formula (XI), the artisan is provided a useful intermediate which can be functionalized to include any number of bifunctional spacers and multifunctional terminal groups which allow the attachment of one, two or more equivalents of biologically active material or leaving groups for later reaction and coupling with biologically active materials, e.g. drugs, small molecules, proteins, peptides, etc.